The long term goal of this research is to characterize the metabolic regulation of pathways of folate metabolism in both normal and diseased states, and to determine reaction mechanisms for folate-dependent enzymes. We propose to continue our studies on methylenetetrahydrofolate reductase, a flavoprotein which regulates a key branch point in folate metabolism. This enzyme catalyzes the first step in the flow of one carbon unit from methylenetetrahydrofolate into the pathway for de novo biosynthesis of methyl groups. We will develop affinity chromatography procedures which are designed to facilitate purification of the enzyme to homogeneity in good yield. We will then determine the physical and catalytic properties of the enzyme, and examine further the exchange of methylene protons associated with reduction of methylenetetrahydrofolate by measuring the isotopic discrimination associated with the exchange reaction. Purification procedures will be developed for the next enzyme in the pathway of methyl group biosynthesis, vitamin B12-dependent methyltetrahydrofolate-homocysteine methyl transferase. We will examine the way in which methyltetrahydrofolate is activated for methyl transfer to the B12 prosthetic group. Particular emphasis will be placed on characterizing interactions between methylenetetrahydrofolate reductase and methyltetrahydrofolate-homocysteine methyl transferase which affect methyl group transfer.